CN115403656A - 一种肠道可吸收的降胆固醇肽及其固相合成方法与应用 - Google Patents
一种肠道可吸收的降胆固醇肽及其固相合成方法与应用 Download PDFInfo
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Abstract
本发明公开了一种肠道可吸收的降胆固醇肽及其固相合成方法与应用。所述降胆固醇的氨基酸序列为His‑Thr‑Ser‑Gly‑Tyr(HTSGY)，其来源于乳清蛋白，是一种肠道可吸收五肽。该多肽能显著降低人体内胆固醇含量，有效的缓解动脉粥样硬化，具有潜在的降低肝胆疾病发病率的功效。该多肽对于开发具有降胆固醇功能的乳制品、动物饲料、保健品、保健食品及药物具有十分重要的意义，具有广泛的应用前景。
Description
技术领域
本发明属于活性肽领域，具体涉及一种肠道可吸收的降胆固醇肽及其固相合成方法与应用。
背景技术
现代医学研究证明，居民的不健康生活作息及不均衡的营养搭配造成了过量胆固醇摄入，高发病率的冠心病、高脂血症和动脉硬化等是由于机体内高浓度的胆固醇水平引起的。我国现阶段治疗的方法主要是采用药物靶向治疗，但其中治疗费用高并且毒副作用大。为了保持健康，最理想的方法是在饮食中补充外源性降胆固醇剂，使机体免受胆固醇的侵害。一些蛋白经过酶解后形成具有降胆固醇活性的小肽，这些小肽可用于开发成降胆固醇类的功能性食品，或成为化学合成药物的替代品。从食品安全性考虑，天然降胆固醇物质具有合成降胆固醇剂无法比拟的优势，已经成为研究热点。
乳清蛋白是某一类蛋白质总称，是经过加工成产干酪而排出的乳清中，含量最高的成分，主要包括β-乳 球 蛋 白（β-lactoglobulin ）、α- 乳 白 蛋 白（α-lactalbumin）、牛血清白蛋白（bovine serum albumin）、乳铁蛋白（lactoferrin）等。乳清蛋白有“蛋白质之王”之称，它不仅具有高蛋白、低脂肪、低胆固醇、消化吸收率高等特点，而且含有人体中20 种必需的脂肪酸，氨基酸组成是最齐全和最合理的。乳清蛋白中还含有很多的功能性成分，如乳铁蛋白、糖巨肽、乳过氧化物酶、生长因子等成分。乳清蛋白经蛋白酶的水解作用能够产生具有特定生物功能特性的乳清蛋白水解物，有研究发现，乳清蛋白水解物具有ACE抑制活性、免疫活性、抗炎症、降血糖和抗氧化等作用，其中有些已经进入工业化生产。
Caco-2细胞是来自于人体的结、直肠癌细胞，在合适的培养条件下能够在聚碳酸酯膜上分化为小肠上皮细胞单层，具有与小肠细胞非常相似的一些性质，如具有细胞极性和紧密连接，有微绒毛结构，存在某些主动转系统，如糖类、氨基酸、二肽等。由于形态和性质同小肠上皮细胞极其相似，Caco-2细胞模型是常用的体外吸收模型，可以以此为基础，有效的研究药物、多肽等物质的吸收。此外，利用Caco-2细胞构建肠道转运模型也常被用来筛选可被肠道完整消化吸收的多肽。
发明内容
针对现有技术的不足，本发明提供了一种肠道可吸收的降胆固醇肽及其固相合成方法与应用，该降胆固醇肽的氨基酸序列为His-Thr-Ser-Gly-Tyr，可被肠道吸收，可用于制备降低胆固醇含量的药物或保健品上的应用，具有良好的市场前景。
为解决现有技术问题，本发明采取的技术方案为：
一种肠道可吸收的降胆固醇肽（HTSGY），所述降胆固醇肽的氨基酸序列为His-Thr-Ser-Gly-Tyr，所述降胆固醇肽可被肠道吸收。
上述肠道可吸收的降胆固醇肽的固相合成方法，将二氯树脂溶胀、洗涤，脱除Fmoc保护基后加入氨基酸进行缩合反应，重复脱除-保护-缩合的过程直至所有氨基酸连接完毕；切割树脂，得到多肽His-Thr-Ser-Gly-Tyr粗品，用反相高效液相色谱法纯化，得到肠道可吸收的降胆固醇肽的纯品。
上述肠道可吸收的降胆固醇肽在制备预防和/或治疗治疗高脂血症或肝胆疾病的药物或保健品上的应用。
上述肠道可吸收的降胆固醇肽在制备预防和/或治疗冠心病药物或保健品上的应用。
有益效果：
与现有技术相比，本发明一种肠道可吸收的降胆固醇肽及其固相合成方法与应用，具有如下优势：
1、本发明的降胆固醇五肽结构清晰明了，可采用固相化学合成方法制得，也可通过对乳清蛋白酶解物进行分离纯化获得，来源广泛，降低了生产成本。
2、本发明的降胆固醇五肽对降低胆固醇含量具有较好的效果，具有潜在的缓解肠道胆固醇吸收的功效，可用于制备高脂血症、冠心病等药物或预防与胆固醇相关疾病发生的保健品，也可与其他保健品或食品添加剂复配使用。
附图说明
图1为本发明所述酪蛋白来源降胆固醇肽制备方法的工艺流程图；
图2为多肽HTSGY（His-Thr-Ser-Gly-Tyr）的色谱图；
图3为多肽HTSGY（His-Thr-Ser-Gly-Tyr）的MS/MS图；
图4 为HTSGY的化学降胆固醇活性。
具体实施方式
以下实施例对本发明作进一步说明，但不理解为对本发明的限制。在不背离本发明精神及实质的情况下，对本发明的方法、步骤或条件所做的修改和替换，均属于本发明的范围。若为特别指出，实施案例中所用的技术手段为本领域技术人员所熟知的常规手段。本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。
实施例1 Caco-2细胞培养
向DMEM培养基（购买于博士德生物）中加入胎牛血清(FBS)，加入量为20％(v/v)，然后加入1％(v/v)由100U/mL青霉素和100μg/mL链霉素组成的混合双抗。细胞培养箱为Caco-2细胞（购于南京凯基生物科技发展有限公司）的生长提供了所需的环境，箱内条件设定为37℃且含有5％的CO2。根据细胞生长情况及时进行换液和传代。
实施例2 His-Thr-Ser-Gly-Tyr多肽的分离纯化方法
（1）制备乳清蛋白酶解液
使用双蒸水配置浓度为6%的乳清蛋白水解液（所用乳清蛋白粉，型号WPC-80，厂家：美国希尔玛乳酪公司），在65℃ 的条件下恒温加热并搅拌，使其充分溶解，用0.5M NaOH和0.5 M HCl溶液调至pH为8.5，将反应装置置于50℃恒温震荡水浴箱中，加入3%糜蛋白酶进行水解，酶解过程中不断加入0.1mol/L的NaOH使之维持溶液pH值恒定，反应时间5h；反应结束后将酪蛋白水解液于90℃ 加热20min灭酶，7000×g离心10min取上清。
（2）制备乳清蛋白肽超滤液
将步骤（1）收集的上清液进行超滤，用截留分子量为10000Da的超滤膜进行超滤，调整超滤膜压力为0.5Mpa(压力不可过大以免损伤仪器)，流速为5-10mL/min左右(流速太快压力大损伤膜结构，太慢则效率低)，得到分子量3kDa以下的超滤多肽溶液，超滤液通过真空冷冻干燥进行浓缩；
（3）制备乳清蛋白源粗肽
将上述浓缩后的乳清蛋白肽超滤液通过凝胶色谱进行分离纯化，称取SephadexG-10固体，用沸水溶胀2h；在层析柱（10*500mm）中加入少量蒸馏水以排除层析柱底部的空气；摇匀溶胀好的Sephadex G-10凝胶，用一根玻璃棒小心将凝胶导入层析柱，柱子装完后用蒸馏水平衡充分；超滤液用蒸馏水溶解，质量浓度为80mg/mL，上样量为3ml，洗脱速度为1.5ml/min，紫外检测器波长为220nm，收集各组分，冷冻干燥。
（4）Caco-2细胞模型模拟小肠吸收
将实施例中培养的Caco-2细胞接种到TransWell小室中，接种密度为2×105个细胞/mL，然后在AP侧（Transwell板小室）加入0.5mL细胞悬液，BL侧（Tranwell板大室）加入1.5mL培养液，放入37℃，5% CO2培养箱内培养。在培养周期第一周隔天换培养液，之后每天换液，培养周期为21天。将步骤（3）中收集的两个峰物质分别用HANKS缓冲液配成20 mg/mL的活性肽溶液，在transwell小室的AP侧加入0.5mL由HANKS缓冲液配制的酪蛋白肽溶液，BL侧加入1.5 mL HANKS缓冲液。将Transwell板放入37℃，5%CO2培养箱中，转运时间为1-2 h。转运结束后，BL侧取出转运样品，进行富集，经0.22μm的水系滤膜过滤后进行冷冻干燥，收集冷冻干燥后的粉末备用。
（5）乳清蛋白源多肽序列分析
选择通过Caco-2细胞转运后的乳清蛋白来源肽通过高效液相质谱联用UPLC-MS分析所含肽序列，UPLC-MS分析条件如下：
液相色谱分析柱：ACQUITY UPLC BEH130 C18柱（2.1×150nm，1.7μm）（Water,美国）；流动相A：100% 乙腈；流动相B：0.1%甲酸水溶液；进样量：8μL；检测波长：204nm；流速：0.3mL/min；柱温：45℃ ；洗脱程序：0~1min，流动相A 2%，流动相B 95%；1~10min，流动相A30%，流动相B 60%；10~17min，流动相A 100%，流动相B 0%；17~22min，流动相A 2%，流动相B95%；
质谱仪采用美国WATERS MALDI SYNAPT Q-TOF MS（Water,美国），干燥气和雾化气采用的是N2。相关的质谱条件为：离子方式：ESI+；毛细管电压：3.5kVolts；锥孔电压：30Volts；脱溶剂气体温度：300℃ ；离子源温度：100℃ ；脱溶剂气体流量：500lit/hr；锥孔气体流量（L/Hr）：50lit/hr；碰撞能量：6/35Volts；质量范围：100～1500m/z；检测电压：1600～1700Volts；利用软件Masslynx4.1进行乳清蛋白肽氨基酸序列分析与手动计算肽的氨基酸序列相结合，确定能完整形式顺利由小肠转运至血液的乳清蛋白肽序列。
图2为多肽HTSGY（His-Thr-Ser-Gly-Tyr）的MS/MS图。
实施例3 固相合成法合成His-Thr-Ser-Gly-Tyr多肽
将二氯树脂溶胀、洗涤，脱除Fmoc保护基后加入氨基酸进行缩合反应，重复脱除-保护-缩合的过程直至所有氨基酸连接完毕。切割树脂，得到多肽His-Thr-Ser-Gly-Tyr粗品，用反相高效液相色谱法纯化，得到多肽HTSGY的纯品(>90％)。
实施例4 五肽LHSMK的化学降胆固醇活性研究
配制胆固醇胶束：10mmol/L牛磺胆酸钠，2mmol/L胆固醇，5mmol/L油酸，135mmol/LNaCl溶解在PBS中的溶液。样品组和空白组分别震荡均匀后超声乳化1 h，37℃恒温放置24h，8000r/min离心10min，取上清液备用。
根据总胆固醇（T-CHO）测试试剂盒（购于南京建成生物工程研究所）的说明测定胆固醇含量，其中OD采用酶标仪进行检测，波长为510nm。
样品组为1mL五肽溶液/和4mL胆固醇胶束；
空白组为1mL蒸馏水和4mL胆固醇胶束；
图4为HTSGY对胆固醇降解能力的测定，其中ALPM、AVFK也是肠道可吸收多肽，但降胆固醇能力明显低于五肽HTSGY。
综上所述，本发明的五肽HTSGY不仅具有降胆固醇能力，还是肠道可吸收多肽，降胆固醇能力高于ALPM和AVFK。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。
Claims (4)
1.一种肠道可吸收的降胆固醇肽，其特征在于，所述降胆固醇肽的氨基酸序列为His-Thr-Ser-Gly-Tyr，所述降胆固醇肽可被肠道吸收。
2.基于权利要求1所述的肠道可吸收的降胆固醇肽的固相合成方法，其特征在于，将二氯树脂溶胀、洗涤，脱除Fmoc保护基后加入氨基酸进行缩合反应，重复脱除-保护-缩合的过程直至所有氨基酸连接完毕；切割树脂，得到多肽His-Thr-Ser-Gly-Tyr粗品，用反相高效液相色谱法纯化，得到肠道可吸收的降胆固醇肽的纯品。
3.基于权利要求1或2所述的一种肠道可吸收的降胆固醇肽在制备预防和/或治疗高脂血症或肝胆疾病的药物或保健品上的应用。
4.基于权利要求1或2所述的一种肠道可吸收的降胆固醇肽在制备预防和/或治疗冠心病药物或保健品上的应用。
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